TY - JOUR
T1 - Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus)
AU - Barlough, Jeffrey E.
AU - Madigan, John E
AU - DeRock, Elfriede
AU - Bigornia, Luisa
PY - 1996/6
Y1 - 1996/6
N2 - A nested polymerase chain reaction for detecting Ehrlichia equi in horses and ticks (Ixodes pacificus) was developed. A major second-round PCR product of 928 bp could be readily visualized in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicated that DNA derived from ≤ 7.6 but > 3 infected neutrophils was sufficient to generate a PCR signal. The specificity of the PCR was examined using a panel of rickettsiae, of which only E. equi and the closely-related human granulocytotropic ehrlichia produced PCR bands. In an in vivo infection study, E. equi DNA was detected in blood buffy-coat cells of an experimentally-infected horse on days three through 14 post- inoculation. In a separate study, three of six adult I. pacificus that as nymphs had been fed on an experimentally infected horse were found to be PCR- positive for E. equi.
AB - A nested polymerase chain reaction for detecting Ehrlichia equi in horses and ticks (Ixodes pacificus) was developed. A major second-round PCR product of 928 bp could be readily visualized in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicated that DNA derived from ≤ 7.6 but > 3 infected neutrophils was sufficient to generate a PCR signal. The specificity of the PCR was examined using a panel of rickettsiae, of which only E. equi and the closely-related human granulocytotropic ehrlichia produced PCR bands. In an in vivo infection study, E. equi DNA was detected in blood buffy-coat cells of an experimentally-infected horse on days three through 14 post- inoculation. In a separate study, three of six adult I. pacificus that as nymphs had been fed on an experimentally infected horse were found to be PCR- positive for E. equi.
KW - Diagnosis Rickettsia
KW - DNA sequence analysis
KW - Ehrlichia equi
KW - Horse
KW - Ixodes pacificus
KW - Polymerase chain reaction
KW - Southern hybridization test
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U2 - 10.1016/0304-4017(95)00904-3
DO - 10.1016/0304-4017(95)00904-3
M3 - Article
C2 - 8966998
AN - SCOPUS:0029934164
VL - 63
SP - 319
EP - 329
JO - Veterinary Parasitology: Regional Studies and Reports
JF - Veterinary Parasitology: Regional Studies and Reports
SN - 0304-4017
IS - 3-4
ER -